173 microorganisms were isolated from 40 infected root canals with refractory apical periodontitis. Among them, Pseudomonas aeruginosa (P.s.), Staphylococcus aureus(S.a.), Bacillus subtilis(B.s.), Enterococcus faecalis(E.t.) and Candida albicans(C.a.) were used. They were adhered to each cell disk and cultured with two kinds of method. One was set in multiplate with TS Broth (method 1), and another was hanged in each small bottle (method 2). As the culture medium of method 2, TS Broth, TS Broth with plasma and saline with plasma were used . Culture periods were 3,7 andl4 days in 37ﾟC.Then each cell disk was observed with SEM.The substances like glycocalyx were observed in each microorganism except E.f. andC.a. (method 1), and except C.a.(method 2), increasing with the extension of culture period. Besides those substances increased in the medium adding plasma.As the medicine against microorganism with biofllm, FC (100%, 10%, 1%), J(100%, 10%, 1%), NaClO(l0%, 5%, 1%) and oxidative potential water were used. After cultivation in TS Broth with plasma for 7 days, each microorganism was immersed in each medicine for 15 minutes and cultured again for 24 and 48 hours. NaClO and oxidative potential water were only used for S.a..Undiluted solutions of FC and J were effective in each microorganism, but diluted solutions reduced the effect. The effect of NaClO also reduced in low concentration. Oxidative potential water was inefficient. Microorganisms can form bioflims in the apical portion of root canal with refractory apical periodontitis. So it seems to be difficult to supply sufficient medicine. In future it will be necessary to study about the method for destruction or melting of bioflims.